Visualization of RNA in living cells

ABSTRACT

A method for visualizing the location and movement of a specific RNA of interest in a living cell, in real time, is disclosed. The method includes the following steps: (a) providing a DNA encoding the RNA, which RNA includes a protein-binding site; (b) providing a nucleic acid encoding a fusion protein, which fusion protein comprises a fluorescent domain and an RNA-binding domain; (c) introducing the DNA encoding the RNA, and the nucleic acid encoding the fusion protein, into a eukaryotic cell so that the DNA encoding the RNA and the nucleic acid encoding the fusion protein are expressed in the cell; and (d) detecting fluorescence outside the nucleus or inside the nucleus of the cell, with the fluorescence being from the fusion protein bound to the RNA. In some embodiments, the fusion protein also includes an intracellular localization domain.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Work on this invention was supported by NIH Grant Nos. GM 54887 and GM57071. Therefor the government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to cell biology, genetics, recombinant DNAtechnology, fluorescence microscopy, and videography.

BACKGROUND OF THE INVENTION

Messenger RNA localization is a well-documented phenomenon and providesa mechanism by which to generate cell assymetry (St. Johnston, Cell81:161-170 (1995); Glotzer et al., Cell Dev. Biol. 7:357-365 (1996);Steward et al., in mRNA Metabolism and Posttranscriptional GeneRegulation, Wiley-Liss, New York, 127-146). Messenger RNA localizationhas been studied by fluorescence in situ hybridization (FISH) (Long etal., RNA 1:1071-1078 (1995). In situ hybridization, and other methodsthat require fixation of cells, offer good spatial resolution, but areseverely limited in temporal resolution. Thus, while these techniquesare well-suited for determining where RNA goes in living cells, they areunsuited for determining how quickly, or by what route, the RNA travelsto its destination.

A further limitation of FISH methods, is that fixation kills cells.Therefore, those methods are incompatible with cell selection, wherecells must be kept alive to initiate a new cell line.

SUMMARY OF THE INVENTION

We have developed a general method for visualizing the location andmovement of a specific RNA of interest in a living cell, in real time.The method includes the following steps: (a) providing a DNA encodingthe RNA, which RNA includes a protein-binding site; (b) providing anucleic acid encoding a fusion protein that includes a fluorescentdomain and an RNA-binding domain that binds to the protein-binding sitein the RNA; (c) introducing the DNA encoding the RNA, and the nucleicacid encoding the fusion protein, into a eukaryotic cell so that the DNAencoding the RNA and the nucleic acid encoding the fusion protein areexpressed in the cell; and (d) detecting fluorescence in the cell, thefluorescence being from the fusion protein bound to the RNA.

Preferably, the RNA includes a multiplicity of protein-binding siteslocated in the 3′ untranslated region (3′UTR) of the RNA. TheRNA-binding domain can be derived from a bacteriophage MS2 protein, andthe protein-binding site can be a bacteriophage MS2 binding site. Inpreferred embodiments of the invention, the fluorescent domain isderived from green fluorescent protein (GFP). In some embodiments, thefusion protein includes an intracellular localization domain, e.g., anuclear localization signal (NLS) domain or a nuclear export signal(NES) domain. When the fusion protein contains an NLS domain,fluorescence from the fusion protein bound to the RNA is detectedoutside the nucleus.

The DNA encoding the RNA, and the nucleic acid encoding the fusionprotein, can be provided on a single vector or on separate vectors. Insome embodiments of the invention, the cell is a yeast cell. The cellcan contain one or more RNA localization factors, e.g., she geneproducts in a yeast cell.

The invention also provides a method for screening a DNA library todetect a DNA encoding an RNA containing a protein-binding site. Themethod includes providing a eukaryotic test cell. The test cellexpresses a fusion protein containing a fluorescent domain and anRNA-binding domain that binds to a protein-binding site. The methodfurther includes transforming the test cell with a candidate DNA fromthe DNA library; and detecting the fusion protein bound to an RNAcontaining the protein-binding site, if present, by measuringfluorescence. In some embodiments, the fusion protein includes anintracellular localization domain, e.g., a nuclear localization signal(NLS) domain or a nuclear export signal (NES) domain. Preferably, thetest cell does not express an endogenous protein that binds to theprotein-binding site.

The invention also provides a nucleic acid encoding a fusion protein.The fusion protein encoded contains a fluorescent domain and anRNA-binding domain. The fluorescent domain can be derived from GFP or aGFP variant, e.g., blue fluorescent protein (BFP), yellow fluorescentprotein (YFP), or cyan fluorescent protein (CFP). The binding domain canbe derived from a bacteriophage MS2 binding protein. In someembodiments, the fusion protein includes an intracellular localizationdomain, e.g., a nuclear localization signal (NLS) domain or a nuclearexport signal (NES) domain. The invention also includes a vectorcontaining the nucleic acid encoding the fusion protein, and a celltransformed with the vector containing the nucleic acid encoding thefusion protein.

The invention also includes a screening method for identifying acompound that inhibits nuclear RNA export or import. The method includesproviding a eukaryotic test cell that expresses a DNA encoding an RNA,which RNA includes a protein-binding site; and expresses a fusionprotein. The fusion protein includes a fluorescent domain and anRNA-binding domain that binds to the protein-binding site in the RNA.The method further includes contacting the test cell with a candidatecompound, and then detecting a candidate compound-related reduction ofnuclear RNA export or import, if present. In some embodiments of themethod, the RNA-binding domain of the fusion protein and theprotein-binding site in the RNA are derived from viral sequences.

The invention also includes a method for detecting, in real-time, thetranscription of a specific gene. The method includes providing aeukaryotic cell that contains: a DNA encoding an RNA that includes aprotein-binding site, and a nucleic acid encoding a fusion protein. Thefusion protein includes a fluorescent domain and an RNA-binding domainthat binds to the protein-binding site. The method further includesdetecting a focus of fluorescence, the focus being from a multiplicitynascent RNA molecules, each nascent RNA molecule being bound to one ormore fusion protein molecules. In some embodiments, the fusion proteinincludes a nuclear export signal domain. The nucleic acid encoding thefusion protein can be transiently expressed from a vector introducedinto the cell.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application, including definitions will control. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described below. The materials,methods, and examples are illustrative only and not intended to belimiting. Other features and advantages of the invention will beapparent from the detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIGS. 1A-1C. FIG. 1A is a schematic diagram of the NLS-MS2-GFP fusionprotein. FIG. 1B is a schematic diagram of the nucleic acid constructused for expression of an NLS-MS2-GFP fusion protein and an ASH1 lacZreporter mRNA in a yeast system. FIG. 1C is a schematic diagram of thenucleic acid construct used for expression of an NLS-MS2-GFP fusionprotein and an ADHII lacZ reporter mRNA in a yeast system. A lacZ-ASH1reporter RNA was used to demonstrate RNA movement/localization in livingcells. A lacZ-ADHII reporter RNA was used as a negative control forrapid movement and localization. The reporter mRNAs contain six bindingsites for the coat protein of the bacterial phage MS2. The 3′UTRs wereeither from the ASH1 gene, to induce mRNA localization at the yeast budtip, or, from the ADHII gene, as a negative control. In addition, anuclear localization signal (NLS) followed by an HA tag, was introducedat the N-terminus of the fusion protein, so that that only the fusionprotein bound to its target, the reporter RNA, would appear in thecytoplasm.

FIG. 2 is a color photomicrograph showing live cells expressing theNLS-MS2-GFP fusion protein, and the lacZ-ASH1 reporter mRNA, whichcontained six MS2 binding sites. Arrows indicate some of the particles,usually in the yeast bud. Bar=5 μm.

FIG. 3 is a color schematic diagram tracing the path of anRNA-containing particle in a yeast mother cell and bud (43 μm per 240seconds). Particle movement was analyzed in a wildtype yeast strain(K699) expressing both the lacZ-ASH1 reporter RNA and the NLS-MS2-GFPfusion protein. Observation was conducted using epifluorescence andbright field microscopy. A cell with minimal nuclear signal was chosenso as not to obscure the particle. Movement of the particle was recordedwith a video camera linked to a VCR. During the period of observation,the 30-second intervals are represented beginning with the coolestcolors (purple) and proceeding to the hottest colors (red). The particlespent 180 out of 240 seconds in the bud, and about 60 seconds localizedat or near the bud tip.

DETAILED DESCRIPTION OF THE INVENTION

A DNA encoding the visualized RNA can be obtained readily from anysuitable source using conventional recombinant DNA technology asnecessary. The examples provided below involve visualizing RNA moleculesengineered to include a lacZ coding region and a yeast ASH1 3′ UTRcontaining intracellular localization signals. In the examples, the RNAmolecules are visualized in yeast cells. It will be appreciated,however, that the methods of this invention are generally applicable todifferent RNAs and different eukaryotic cells.

The invention can be used to visualize RNAs whose movement depends onparticle formation and RNAs that move without particle formation.Moreover, the invention is useful for visualizing non-localized RNAs, aswell as localized RNAs. In some embodiments, the RNA to be visualizedencodes a separately detectable polypeptide, e.g., β-galactosidase.Choosing or designing an RNA suitable for visualization according tothis invention, and obtaining a DNA encoding the chosen RNA is withinordinary skill in the art.

For some RNAs in some cell types, RNA localization may be partially orcompletely dependent upon trans-acting localization factors. Therefore,in some embodiments of the invention, localization factors are presentin the living cells in which the RNA is visualized. Localization factorsare exemplified by the products of the she genes in yeast. These arecytoplasmic factors. Nuclear factors exporting the RNA are present inall eukaryotic cells.

The RNA to be visualized contains at least one, and preferably amultiplicity of protein-binding sites. In some embodiments of theinvention, from 5 to 10 sites are suitable. In other embodiments, from10 to 50 binding sites will be suitable. In yet other embodiments, morethan 50 binding sites, i.e., up to several hundred, may be desirable. Ingeneral, increasing the number of binding sites in the RNA increasesfluorescence signal strength due to an increased number of fluorescentdomains bound per RNA molecule. Some RNAs already contain suitableprotein-binding sites. DNAs encoding such RNAs can be used in thisinvention without incorporation of an exogenous protein-binding site.When the RNA to be visualized does not already contain a suitableprotein-binding site, a suitable exogenous protein-binding site isincorporated into a DNA encoding the RNA.

The protein-binding site is a nucleotide sequence. Preferably, a singleprotein-binding site consists of a single, contiguous region of RNA,e.g., a stem-loop structure. Preferably, the length of the single,contiguous region of RNA is less than 100 nucleotides, more preferablyit is less than 50 nucleotides, and most preferably, it is between 15and 25 nucleotides. Preferably, the binding interaction between theprotein-binding site and the binding domain displays high specificity,which results in a high signal-to-noise ratio.

A preferred protein-binding site is the bacteriophage MS2 binding site.Complete MS2 nucleotide sequence information can be found in Fiers etal., Nature 260:500-507 (1976). Additional information concerning theMS2 sequence-specific protein-RNA binding interaction appears inValegard et al., J. Mol. Biol. 270:724-738 (1997); Fouts et al., NucleicAcids Res. 25:4464-4473 (1997); and Sengupta et al., Proc. Natl. Acad.Sci. USA 93:8496-8501 (1996).

Other binding site/binding domain pairs can be used instead of theMS2-derived pair. A second useful binding site/binding domain pair isthe hairpin II of the U1 small nuclear RNA and the RNA-binding domain ofthe U1A spliceosomal protein (Oubridge et al., Nature 372:432-438(1994). A third useful alternative binding site/binding domain pair isthe protein IRP1 and its RNA target, the IRE (Klausner et al., Cell72:19-28 (1993); Melefors et al., Bioessays 15:85-90 (1993). The IRE isa stem-loop structure found in the untranslated regions of mRNAsencoding certain proteins involved in iron utilization, and it bindsspecifically to IRP1. A fourth useful alternative binding site/bindingdomain pair is HIV REV and RRF. A fifth useful alternative bindingsite/binding domain pair is a zipcode binding protein and a zipcode RNAelement (Steward et al, supra). A sixth useful alternative bindingsite/binding domain pair is a box C/D motif and box C/D snoRNAfamily-specific binding protein (Samarsky et al., EMBO J. 17:3747-3757(1998).

In addition, the protein-binding site can be an aptamer produced by invitro selection. An aptamer that binds to a protein (or binding domain)of choice can be produced using conventional techniques, without undueexperimentation. Examples of publications containing useful informationon in vitro selection of aptamers include the following: Klug et al.,Mol. Biol. Reports 20:97-107 (1994); Wallis et al., Chem. Biol.2:543-552 (1995); Ellington, Curr. Biol. 4:427-429 (1994); Lato et al.,Chem. Biol. 2:291-303 (1995); Conrad et al., Mol. Div. 1:69-78 (1995);and Uphoff et al., Curr. Opin. Struct. Biol. 6:281-287 (1996).

In some embodiments of the invention, the protein-binding site(s) is(are) located in the 3′UTR of the RNA to be visualized. However, thelocation of the protein-binding site(s) can be elsewhere in the RNAmolecule. The RNA can contain other genetic elements, e.g., one or moreintrons, stop codons, and transcription terminators. In addition, theDNA encoding the RNA contains a promoter operably linked to thetranscribed region.

The invention utilizes a fusion protein that includes at least twodomains. One domain is a fluorescent domain. A preferred fluorescentdomain is derived from a GFP. Naturally-occurring GFPs causebioluminescence, e.g., in the jellyfish Aequorea victoria. In GFPs,fluorescence is produced by the interaction of modified amino acids inthe GFP polypeptide chain. Formation of the GFP fluorophore is speciesindependent, but GFPs can be modified through mutagenesis to optimizetheir function in different species. See, e.g., Cubitt et al.,“Understanding, improving and using green fluorescent proteins,” Trends.Biochem. Sci. 20:488-455 (1995). When a GFP embodiment of the inventionis used in yeast, preferably the GFP domain of the fusion protein isencoded by a yeast-optimized version of a GFP cDNA. See, e.g., Cormacket al., Microbiology 143:303-311 (1997). Variants of GFP include bluefluorescent protein (BFP), yellow fluorescent protein (YFP), and cyanfluorescent protein (CFP).

The second domain of the fusion protein is an RNA-binding domain. Thisdomain recognizes and interacts with the protein-binding site (in theRNA discussed above) in a specific binding interaction, underphysiological conditions. A preferred RNA-binding domain is derived fromthe bacteriophage MS2 coat protein (capsid), which binds with highspecificity to a unique site on MS2 RNA. See Fiers et al. (supra),Valegard et al. (supra), Fouts et al. (supra); and Sengupta et al.,(supra). Other proteins containing RNA-binding domains are discussedabove.

In some embodiments, the fusion protein includes a third domain thatcauses intracellular localization of the fusion protein when the fusionprotein is not bound to its RNA target. Examples of intracellularlocalization domains include nuclear localization signal (NLS) domains,nuclear export signal (NES) domains, and nucleolar targetting domains.Various NLS sequences are known in the art, any of which can be used inthe invention. A preferred NLS is derived from SV40, a well-known simianvirus. A useful NES can be derived from human immunodeficiency virus(HIV) REV sequences.

When the fusion protein contains an NES domain, fluorescence is detectedinside the nucleus where the protein is bound to its target RNA. Incases where the RNA is undergoing biosynthesis at the site of its gene(transcription site), the fluorescence is most intense at thetranscription site. Thus, the site of active transcription of a specificgene in a living cell is visualized.

A fusion protein containing an REV-derived NES can be used as a tool toevaluate therapeutic agents that interdict HIV export. To do so, onemeasures flow of the REV-derived NES-containing fusion protein out ofthe nucleus, in the presence of a reporter RNA containing theprotein-binding site known as “RRE” (REV-Responsive Element).

If the fusion protein does not include an intracellular localizationdomain, or if it contains an NLS, binding of the fusion protein to itsRNA target can be indicated by a low fluorescence level in the nucleus(relative to the fluorescence level in the cytoplasm), when the RNA isin excess. In this situation, decreased nuclear fluorescence resultsfrom the fusion protein being dragged out of the nucleus by exiting RNAto which the protein is bound. Decreased nuclear or cytoplasmicfluorescence may be advantageous when the fusion protein is employed incells subjected to flow sorting.

When the RNA includes a C/D box as well as a protein-binding siterecognized by the fusion protein's binding domain, the RNA and fusionprotein can be used in assays to screen for drugs that affectintranuclear targeting or nucleolar function.

DNAs useful to encode and express the reporter RNA (containing one ormore protein-binding sites) and the fusion protein are constructed usingconventional recombinant DNA techniques. Such techniques are well knownin the art, and can be found in standard references such as thefollowing: Sambrook et al., Molecular Cloning—A Laboratory Manual (2ndEd.), Cold Spring Harbor Laboratory Press (1989); Innis et al. (eds.)PCR Protocols—A Guide to Methods and Applications, Academic Press, SanDiego, Calif. (1990); Perkin-Elmer manual for PCR. Numerous DNAscontaining useful coding sequences, expression control sequences, andrestriction endonuclease sites to facilitate manipulation, arecommercially available.

The DNA sequences required for expression of the RNA to be visualized(reporter RNA), and expression of the fusion protein, can beincorporated into a single vector. Preferably, however, the sequencesare incorporated into two separate vectors. Vectors used in theinvention are selected for compatibility with the cells in which theywill be used. Expression vectors designed for use in particular celltypes, with convenient restriction sites to facilitate the cloning ofinserts, are commercially available and can be used in the invention.Preferably, the promoters used to drive expression of the RNA and thefusion protein are chosen so that the RNA expressed is in excessrelative to the fusion protein.

In the practice of this invention, fluorescence microscopy imageacquisition and processing can be carried out using conventional opticalsystems, computer hardware, and software. Image acquisition systems foruse in the invention can be devised by the skilled person or obtainedcommercially. Suitable image capture software includes CellSCAN™software (Scanalytics, Fairfax, Va.). Similarly, video data capture andprocessing can be carried out using convention hardware and software.Software useful with video data includes NIH Image (National Institutesof Health, Bethesda, Md.). It is envisioned that automated scanningprocedures can be used with this invention, including microwell platereaders and flow cytometers.

Automation, however, is not required. Microscopic visual analyses willalways be feasible.

The methods described here for visualizing RNA movement in living cellsare generally applicable to the investigation of any RNA-proteincomplex, such as those involved in RNA processing, nuclear export, orintranuclear targeting.

The invention is further illustrated by the following experimentalexamples. The examples are provided for illustration purposes only, andthey are not to be construed as limiting the scope or content of theinvention in any way.

EXAMPLES

A two-plasmid system was constructed and tested successfully in yeast.In one plasmid, a GFP cDNA sequence was fused to coding sequences forthe single-stranded RNA phage capsid protein MS2 (Fouts et al., supra).A nuclear-localization signal was engineered into the fusion protein(FIG. 1A). This caused the fusion protein to be restricted to thenucleus if not complexed to RNA. The fusion protein was expressed fromthe strong constitutive GPD promoter (Schena et al., “Guide to yeastgenetics and molecular biology,” in Methods in Enzymology, Gutherie etal., eds., Academic Press, New York (1991), pp. 389-398).

The second plasmid encoded a reporter RNA containing an ASH1 mRNA 3′UTRfused to a lacZ coding region (Long et al, Science 277:383-387 (1997)).Six MS2 binding sites, each consisting of a 19 nucleotide RNA stem-loop(Valegard et al, supra) were inserted downstream of the lacZ codingregion (FIG. 1B). The cluster of MS2 binding sites provided foramplification of the GFP fluorescence signal due to binding of up to sixfusion proteins, each containing a GFP domain. Transcription of thereporter RNA was under control of a galactose inducible promoter, asdescribed in Long et al., RNA 1:1071-1078 (1995)).

Yeast cells expressing both the GFP-MS2 chimera and the ASH1 reportercontained a single, bright “particle” that was usually localized at thebud tip (FIG. 2).

To confirm that particle formation and localization were dependent onthe ASH1 3′UTR in the reporter RNA, the ADHII 3′UTR was substituted inplace of the ASH1 3′UTR. It was known that the ADHII 3′UTR sequence wasunable to localize a reporter RNA to the bud tip. When the fusionprotein was co-expressed with the RNA containing the ADHII 3′UTR, GFPfluorescence was diffuse, throughout the cytoplasm.

To confirm that the brightly fluorescent particles represented from theNLS-MS2-GFP fusion protein bound to the lacZ-ASH1 reporter RNA,fluorescence in situ hybridization was performed, using probes specificfor lacZ. In cells expressing the lacZ-ASH1 reporter, the reporter mRNAcolocalized with the fusion protein in the particle. When the lacZ-ADHIIreporter RNA was used, in situ hybridization to lacZ sequences showed adiffuse reporter RNA distribution, which colocalized with thefluorescence signal from the fusion protein. When the fusion protein wasexpressed in cells without any reporter mRNA present, or co-expressedwith an RNA lacking MS2 binding sites, GFP fluorescence was mainlyrestricted to the nucleus.

To determine whether the fusion protein artifactually induced particleformation, the MS2 binding sites were deleted from the lacZ-ASH1reporter. In situ hybridization (following galactose induction) usinglacZ probes revealed reporter RNA concentrated in a particles in manycells. The fusion protein was not similarly concentrated.

In contrast to the single particles observed with the lacZ-ASH1 reporterRNA, endogenous yeast ASH1 mRNA localized in a number of spots forming acrescent at the bud tip (detected by in situ hybridization).

In control cells, e.g., cells without reporter RNA, dim GFP signals wereseen occasionally. These dim signals were not scored as particlesbecause of their relative dimness, and because they never localized inthe bud. The fluorescence intensity of the dim signals was approximatelyan order of magnitude below that of the particles formed in the presenceof the ASH1 3′UTR. These dim signals may have represented aggregation ofthe fusion protein, even though we used a mutant version of MS2 reportedto be deficient in self-assembly (Lim et al., Nucl. Acids Res.22:3748-3752 (1994)).

Experimental results indicated that the ASH1 3′UTR facilitated theformation of a multi-molecular RNA particle. These particles wereobserved in the mother cell, and occasionally they were seen moving fromthe mother to the daughter cell. Therefore, they were deemed likely tobe the vehicle by which ASH1 mRNA normally localizes in yeast. Becauseof the bright particles, localization was easily determined.

Yeast she mutants were known to be defective in ASH1 RNA localization(Long et al., 1997, supra; Takizawa et al., Nature 389:90-93 (1997)).Therefore, we tested she mutants for particle localization. In the shemutant strains, the number of particles was significantly decreasedcompared to the wild-type. The relatively few particles that formedfailed to localize. In a she 5 mutant strain, the particle stayed at thebud neck. In a she3 mutant strain, the single, bright particlesdispersed into many smaller particles, none of which localized. In ashe1/myo4 mutant strain, particles which formed stayed in the mother. Ina she2 mutant strain, particles were almost completely absent. Thisconfirmed that particle observation was a surrogate assay for RNAlocalization.

Since the particles were bright enough to be followed in living cells,we observed their movement in real time, using video microscopy. Thisallowed us to ascertain whether the myosin directly transported theparticle from mother to daughter cells. When a moving particle wasidentified, its movement was analyzed for up to four minutes. Movementwas observed in about half of the wild type cells.

Although most of the particles were localized at or near the bud tip,they were occasionally observed moving from the mother cell to the bud.This movement sometimes occurred bidirectionally, with the particlereversing toward (but not into) the mother, and then back to the budtip. In the mother cell, sometimes the particle moved around randomlyand then accelerated through the bud neck, where velocity was thehighest (net displacement per unit of time). Once in the bud, theparticle moved in the distal region and occasionally stalled at the budtip for periods exceeding one minute.

Movement of one of the wildtype particles traveling from mother cell tobud was analyzed in detail. The movement was generally directional, butthe particle wandered over a path five times longer than the shortestpossible distance to the bud tip. This travel path is shown in FIG. 3.The particle moved at velocities varying between 200 and 440 nm/sec(averaged over a moving window of 3 seconds). The localization time(mother to bud tip) for the particle was 128 seconds. Because of theshort time required for localization, the movement of the RNA was a rareevent in the steady-state population. This short time for RNA transportemphasized the importance of using living cells to investigate theprocess of localization.

The visualization of RNA movement in a living cell presented a dynamicview of the localization mechanism. Several insights into thelocalization process resulted from this approach. A first insight wasthat RNA transport occurred via a macromolecular complex, a particle. Asecond insight was that the speed of movement of the RNA-containingparticle was such that it moved to its destination within a few minutes.A third insight was that genes required for localization appear tointeract with the RNA via the particle.

The transport of the reporter could be visualized because of theformation of a particle. Because the particle formation was dependent onspecific sequences in the ASH1 3′UTR sufficient for directing a LacZreporter RNA to the bud, and because it could not localize in she mutantstrains, the particle served as a reporter for localization. Sheproteins and sequences from the ASH1 mRNA 3′UTR participated in particleformation. The particle may have been directly associated with myosin,possibly through She3p.

Yeast Genotypes

The following yeast genotypes were used in the experiments describedhere.

wild type: k699 genotype: (Matα, his3-11, leu2-3, ade2-1, trp1-1, ura3,ho, can1-100)

she1: K5209 genotype (Matα, his3, leu2, ade2, trp1, ura3, can1-100,she1::URA3)

she2: K5547 genotype (Matα, his3, leu2, ade2, trp1, ura3, HO-ADE2,HO-CAN1, she2::URA3)

she3: K5235 genotype (Matα, _his3, leu2, ade2, trp1, ura3, can1-100,she3::URA3)

she4: K5560 genotype (Matα, his3, leu2, ade2, trp1, ura3, she4::URA3)

she5: K5205 genotype (Matα, his3, leu2, ade2, trp1, ura3, can1-100,she5::URA3)

Reporters containing MS2 binding sites

Two repeats of a high affinity MS2 binding site was amplified by PCRfrom the pIIIA-MS2-2 plasmid (Sengupta et al., supra) with the followingoligonucleotides:

5′ CTAGCTGGATCCTAAGGTACCTAATTGCCTAGAAAACATGAGGA (SEQ ID NO: 1), and

5′ ATGCTAAGATCTAATGAACCCGGGAATACTGCAGACATGGGAGAT (SEQ ID NO:2).

The PCR product was digested with BamHI and BglII, and self-ligated inpresence of the same enzyme, to multimerize the MS2 sites inhead-to-tail orientation. The DNA corresponding to a six-repeat of theMS2 site was gel purified, and ligated into the BamHI and BglII sites ofpSL1180 (Pharmacia), to give the plasmid pSL-MS2-6. The plasmids pXR55(ASH1 3′UTR) and pXR2(ADHII 3′UTR) were generated (respectively) bysubcloning the lacZ-ASH1 3′ UTR and the lacZ-ADHII reporter constructsinto the yeast vector YEplac195 (Gietz et al., Gene 74:527-534 (1988))as a PstI/EcoRI restriction fragment generated by PCR and DNArestriction digests. The lacZ-ASH1 3′ UTR cassette originated fromplasmid pXMRS25. The lacZ-ADHII cassette originated from plasmidpHZ18-polyA (Long et al., RNA 1:1071-1078 (1995); Long et al., 1997(supra). Both pXR55 and pXR2 contained the URA3 selectable marker andthe 2 micron origin of replication, and expressed the reporter mRNAsfrom a galactose inducible promoter. Plasmid pSL-MS2-6 was digested byMscI and EcoRV, and cloned at the KpnI site of pXR55, to givepGal-lacZ-MS2-ASH1/URA. Alternatively, it was digested with BamHI andNheI and cloned between the BglII and XbaI sites andpGal-lacZ-ADHII/URA, to yield plasmid pGal-lacZ-MS2-ADHII/URA. TheGAL-lacZ-MS2-ASH1 reporter cassette was then moved into YEplac 112, TRP1selectable, 2 micron plasmid by cloning the ScaI-EcoRI fragment ofpGAL-lacZ-MS2-ASH1/URA into the ScaI and EcoRI sites of pYEplac112, togive pGAL-lacZ-MS2-ASH1/TRP.

Fusion protein vector construction

A yeast-optimized version of the GFP cDNA (Cormack et al., supra) wasamplified by PCR with the following oligonucleotide primers:

5′ GTATCAGCGGCCGCTTCTAAAGGTGAAGAATTA (SEQ ID NO: 3)(yGFP/5′), and

5′ TGACCTGTCGACTTTGTACAATTCATCCAT (SEQ ID NO: 4)(yGFP/3′).

The resulting PCR product was then digested with NotI.

A plasmid containing the HA-tagged MS2 mutant protein dlFG was obtainedfrom Philippe Couttet (IJM, Paris). This cDNA was PCR amplified with thefollowing oligonucleotides:

5′ TCAGTCGCGGCCGCGTAGATGCCGGAGTTT (MS2/3′)(SEQ ID NO: 5), and

5′ TAGCATGGATCCACCATGCCAAAAAAGAAAAGAAAAAGTTGGCTACCCCTACGACGTG CCCGA(NLS-MS2/5′) (SEQ ID NO: 6).

The translation start codon (ATG) followed by the SV40 nuclearlocalization sequence is indicated by underlining. The resulting PCRproduct was then digested with NotI. The two PCR products were thenligated, and the GFP-MS2 chimeric cDNA was reamplified with the GFP/3′and the NLS-MS2/5′ oligonucleotides. The resulting PCR product was thendigested with BamHI and SalI, and ligated into the corresponding site ofthe LEU2 selectable, 2 micron pG14 plasmid (Lesser et al., Genetics133:851-863 (1993); gift of J. Warner) to give pGFP-MS2/LEU.

Fusion protein expression

The strain K699 (Matα, trp1-1, leu2-3, his3-11, ura3, ade2-1, ho,can1-100) was transformed with various combinations of the episomalvectors described above and below, and selected on the appropriateselection media to maintain the plasmids. Yeast cells were then grown tomid-log phase in synthetic media containing 2% raffinose. Cells weresubsequently induced with 3% galactose for 3 hours or the indicatedtimes, to induce expression of the reporter mRNA. Due to the variableexpression levels of the two plasmids, some cells had particles withoutmuch GFP nuclear signal, while other cells had strong GFP signal withoutvisible particles.

Measurement of particle brightness

Particles displayed a range of intensities. The single, bright particlesin cells with the ASH1 reporter, and the much weaker particulate signalsin the control cells, were digitally imaged, using Cellscan software(Scanalytics, Va.). Total fluorescence intensity was measured. Thesingle, bright particle had a fluorescent intensity 10.7 times brighterthan the relatively dim particulate signals observed in controls. Thus,particle signals and non-particle signals were easily distinguished.

Particle counts were scored by three individuals. In different isolatesof the wildtype strain, fixed cells containing the bright, singleparticles ranged from 54% to 68%. Localization in these cells rangedfrom 64% to 78%.

In situ hybridization

Yeast cells were processed for in situ hybridization essentially asdescribed in Long et al., 1995 (supra); and Long et al., 1997 (supra),except that the hybridization mixture and the wash solutions containedonly 10% formamide. The oligo-Cy3-conjugated probes were also asdescribed in Long et al., 1995 and Long et al., 1997.

Cells were prepared for immunofluorescence as for in situ hybridization.After permeabilization overnight in 70% ethanol, the cells wererehydrated in antibody buffer (2×SSC, 8% formamide) for 10 minutes atroom temperature, and then incubated in antibody buffer containing 0.2%RNAse DNAse free BSA and an anti-myc antibody (gift from K. Nasmyth)diluted 1:5, for 1 hour at 37° C. Cells were then washed for 30 minutesat room temperature in antibody buffer, and further incubated for 1 hourat 37° C. with a Cy3 conjugated anti-mouse secondary antibody diluted1:700, in antibody buffer. Cells were mounted in mounting media asdescribed in Long et al., 1996 (supra) and Long et al., 1997 (supra),after a final 30-minute wash at room temperature, in antibody buffer.

Image acquisition and processing

Images were captured using CellSCAN software (Scanalytics, Fairfax, Va.)on an Optiplex GXpro computer (Dell, Austin, Tex.) with a CH-250 16-bit,cooled CCD camera (Photometrics, Tuscon, Ariz.) mounted on a Provis AX70fluorescence microscope (Olympus, Melville, N.Y.) with a PlanApo 60x,1.4 NA objective (Olympus) and HiQ bandpass filters (Chroma Technology,Brattleboro, Vt.). The fluorescence illumination was controlled by thesoftware using a Uniblitz VS25 shutter (Vincent Associates, Rochester,N.Y.). When images were restored, a three-dimensional data set, composedof 20-25 images separated by 200 nm in the axial direction, was acquiredand deconvolved with an acquired point spread function (PSF) using EPRsoftware (Scanalytics). The software controlled the axial position ofthe objective using a PZ54 E piezoelectric translator (PhysikInstrumente, Costa Mesa, Calif.). The PSF is a data set, composed of40-50 images separated by 200 nm in the axial direction, of afluorescent microsphere (Molecular Probes, Eugene, Oreg.) that was 200nm in diameter. A single median plane was recorded for blue filteredimages.

Colocalization of GFP particle and She1/MYo4myc

Twenty cells were analyzed for colocalization of the particle andShe1myc. Thirty optical sections of each cell were taken, and the redand green images were superimposed for each image plane. The number ofparticles colocalizing with She1myc in the same plane were counted.About half (45%) were colocalized with She1myc. For a control, wild typecells containing GFP particles without She1myc were evaluated byidentical methods. No colocalization was observed with theimmunofluorescent antibodies.

Cloning and Epitope Tagging of SHE Genes

The SHE genes were isolated from yeast genomic DNA (strain K699) by PCR.Primers were designed to obtain a PCR fragment of the respective SHEgene to include 1 kb of the promoter as well as 1 Kb of the 3′-UTRregion. The cloned SHE genes were subcloned into YEplac112 and YCplac22and transformed into yeast strains disrupted for the respective gene andtested for functionality by rescue of ASH1 mRNA localization asdetermined using fluorescence in situ hybridization.

A unique restriction site was introduced after the corresponding startcodon or in front of the respective stop codon for each of the SHE genes(except SHE1) using a splicing through overlap extension strategy. Fourprimers were designed for each SHE gene. This gave rise to two PCRfragments that had an overlapping sequence containing a singlerestriction site at the N-terminal or C-terminal part of the gene. Thesefragments served as template in a second PCR step used to amplify thefinal fragment, which was cloned in YEplac112.

Three Myc epitopes were introduced in these unique sites using BamHI (inthe case of SHE3) or XbaI (SHE2) fragments of a Myc epitope cassette(from plasmid pC3003, gift from K. Nasmyth). In the case of SHE1/MYO4, aC-terminal SpeI site (25 amino acids upstream from the stop codon) wasused to subclone a SpeI fragment of a c-Myc epitope cassette (fromplasmid pC3390, gift from K. Nasmyth) containing nine Myc epitopes.Expression of the Myc-epitope tagged proteins was also shown by Westernblots.

After demonstrating functionality, SHE-myc plasmids were transformedinto K699, containing a wildtype locus for each SHE gene with theGFP-reporter plasmids for colocalization studies.

Video analysis

Live cells were mounted between two coverslips and visualized on aninverted microscope (Nikon, Melville, N.Y.) with a PlanApo 60x, 1.4 NA,Ph4 objective (Nikon) using simultaneous brightfield and epifluorescenceillumination. Live video was captured using a C2400 Silicon IntensifiedTube Camera (Hamamatsu, Oakbrook, Ill.) with a 2× eyepiece and recordedon video tape in S-VHS format. Appropriate sequences from the tape weredigitized at a rate of one frame per second using NIH Image software(NIH, Bethesda, Md.) with a frame size of 640×480 pixels on a PowerMacintosh 7600 computer (Apple, Cupertino, Calif.) with S-Videointerface. Using NIH Image, the particle's position in each capturedframe was tabulated and then used to calculate distance travelled andspeed.

Motor analysis

Four minutes of video analysis of a specific particle moving from mothercell to bud was analyzed at one-second intervals. The movement was thenaveraged over a moving time window of three-second time points, andspatially filtered to require a total net travel of five pixels (about0.5 μm) during this time window. The wild-type movement throughout thetime frame resulted in 15 of these 3-second ‘jumps’ at intervals rangingfrom 5 to 30 seconds.

Distances moved during the time frame ranged from 0.6 to 1.4 μm. Averagespeeds per ‘jump’ ranged from 200 to 440 nm/sec. The she1 deletionstrain showed no movement when subjected to this spatial filtering.Effectively, this approach subtracted the background she1 movement fromthe wildtype to reveal the motility characteristics of the She1/Myo4p.

Visualization of RNA not incorporated into particles

The bright particle observed in yeast may not be present in cells otherthan yeast cell, e.g., in vertibrate cells. RNA not incorporated intoparticles is visualized as follows. Forty-eight MS2 binding sites areincorporated into an actin mRNA. When all the MS2 binding sites areloaded with, i.e., bound to, a fusion protein containing a GFP domainand an MS2 binding domain, a single mRNA molecule is visually detectableby GFP fluorescence, using techniques such as those described in Feminoet al., Science 280:585-590 (1998).

Other embodiments are within the following claims.

6 1 44 DNA Bacteriophage MS2 1 ctagctggat cctaaggtac ctaattgcctagaaaacatg agga 44 2 45 DNA Bacteriophage MS2 2 atgctaagat ctaatgaacccgggaatact gcagacatgg gagat 45 3 33 DNA Unknown most probably a yeast 3gtatcagcgg ccgcttctaa aggtgaagaa tta 33 4 30 DNA Unknown most probably ayeast 4 tgacctgtcg actttgtaca attcatccat 30 5 30 DNA Unknown mostprobably a yeast 5 tcagtcgcgg ccgcgtagat gccggagttt 30 6 63 DNA Unknownmost probably a yeast 6 tagcatggat ccaccatgcc aaaaaagaaa agaaaaagttggctacccct acgacgtgcc 60 cga 63

We claim:
 1. A method for visualizing an RNA in a living cell,comprising: providing a DNA encoding the RNA, which RNA comprises anaturally-occurring protein-binding sequence or an engineeredprotein-binding sequence; providing a nucleic acid encoding a fusionprotein, which fusion protein comprises a fluorescent domain and anRNA-binding domain that binds to the protein-binding sequence;introducing the DNA encoding the RNA, and the nucleic acid encoding thefusion protein, into a eukaryotic cell so that the DNA and the nucleicacid are expressed in the cell; and detecting fluorescence in the cell,the fluorescence being from the fusion protein bound to the RNA; therebyvisualizing the RNA in the living cell.
 2. The method of claim 1,wherein the fusion protein further comprises a domain that causesintracellular localization of the fusion protein when the fusion proteinis not bound to its RNA target.
 3. The method of claim 1, wherein theRNA comprises a multiplicity of protein-binding sequences.
 4. The methodof claim 1, wherein the protein-binding sequence is located in the 3′untranslated region of the RNA.
 5. The method of claim 1, wherein theRNA-binding domain is derived from an MS2 protein, and theprotein-binding sequence is a bacteriophage MS2 binding site.
 6. Themethod of claim 1, wherein the fluorescent domain is derived from greenfluorescent protein.
 7. The method of claim 1, wherein the DNA encodingthe RNA, and the nucleic acid encoding the fusion protein, are providedon separate DNA vectors.
 8. The method of claim 1, wherein the cellcomprises a trans-acting localization factor that affects localizationof the RNA.
 9. The method of claim 1, wherein the cell is a yeast cell.10. The method of claim 2, wherein the intracellular localization domainis selected from the group consisting of a nuclear localization signaldomain and a nuclear export signal domain.
 11. The method of claim 10,wherein the intracellular localization domain is a nuclear localizationsignal domain, and the fluorescence from the fusion protein bound to theRNA is detected outside the nucleus.
 12. A method for screening a DNAlibrary to detect a DNA encoding an RNA comprising a protein-bindingsequence, the method comprising: providing a eukaryotic cell thatexpresses a fusion protein comprising a fluorescent domain and anRNA-binding domain that binds to a protein binding sequence;transforming the test cell with a candidate DNA from the DNA library;and detecting the fusion protein bound to an RNA comprising theprotein-binding sequence, if present, by measuring fluorescence, therebydetecting an RNA comprising the protein-binding sequence.
 13. The methodof claim 12 wherein the test cell does not express an endogenous proteinthat binds to the protein-binding sequence.
 14. The method of claim 12,wherein the DNA encoding the RNA comprises a multiplicity ofprotein-binding sequences.
 15. The method of claim 12, wherein theRNA-binding domain is derived from a bacteriophage MS2 protein, and theprotein-binding sequence is a bacteriophage MS2 binding site.
 16. Themethod of claim 12, wherein the fluorescent domain is derived from greenfluorescent protein.
 17. The method of claim 12, wherein the fusionprotein further comprises an intracellular localization domain.
 18. Ascreening method for identifying a compound that inhibits nuclear RNAexport or import, the method comprising: providing a eukaryotic testcell that: (a) expresses a DNA encoding an RNA, which RNA comprises aprotein-binding sequence; and (b) expresses a fusion protein comprisinga fluorescent domain and an RNA-binding domain that binds to theprotein-binding sequence in the RNA; contacting the test cell with acandidate compound; detecting a candidate compound-related reduction ofnuclear RNA export or import, if present; thereby identifying a compoundthat inhibits nuclear RNA export or import.
 19. The method of claim 18,wherein the RNA-binding domain of the fusion protein and theprotein-binding sequence of the RNA are derived from viral sequencesthat encode a viral RNA binding protein and an RNA sequence that isrecognized and bound by that viral RNA binding protein.
 20. A method fordetecting, in real-time, the transcription of a specific gene,comprising: providing a eukaryotic cell that comprises: (a) a DNAencoding an RNA that comprises a protein-binding sequence, and (b) anucleic acid encoding a fusion protein, which fusion protein comprises afluorescent domain and an RNA-binding domain that binds to theprotein-binding sequence; detecting a focus of fluorescence, the focusbeing from a multiplicity of nascent RNA molecules, each nascent RNAmolecule being bound to one or more fusion protein molecules; therebydetecting, in real time, the transcription of a specific gene.
 21. Themethod of claim 20, wherein fusion protein includes a nuclear exportsignal domain.
 22. The method of claim 20, wherein the nucleic acidencoding the fusion protein is transiently expressed from a vectorintroduced into the cell.